Individuals who carry mutations in the breast cancer susceptibility gene, BRCA1, are predisposed to early onset breast and ovarian cancer, some of the most common malignancies in Western societies. Such mutations in BRCA1 account for almost all families with inherited breast and ovarian cancer and for approximately half of families with breast cancer only. The detection of loss-of-heterozygosity (LOH) affecting the wild-type BRCA1 allele in tumors from BRCA1 carriers implies that BRCA1 is a tumor suppressor. The involvement of BRCA1 in breast cancer is complex; to date, more than 100 unique, naturally occurring BRCA1 germline mutations have been identified. The study of BRCA1 has important implications for breast cancer research and attempts to elucidate its biochemical function have included identifying its protein partners, such as BAP1. BAP1 is a member of the UCH family of deubiquitinating enzymes (DUB). These are proteases that reverse the conjugation of ubiquitin to proteins targeted for degradation by the proteasome or relocalization in response to ubiquitination. The conjugation of ubiquitin has been shown to be important in control of many regulatory pathways including; cell cycle regulation, chromatin structure, DNA repair and genome stability, transcription, viral pathogenesis, immune response, and protein quality control. Deubiquitinating enzymes are likely to be useful drug targets in at least some pathological conditions. With the exception of neuronal UCH-L1, no DUB has been the target of a systematic screen. We have developed the means to produce significant amounts of a generic DUB substrate at affordable costs and have chosen to focus our efforts first on a DUB associated with the BRCA1 tumor suppressor. We have begun to optimize the screening conditions and have shown that the assay is adaptable to the 384 well format. We will mount a conventional high throughput drug screen of available NIH compound libraries to identify inhibitors and activators of DUB action. This screen will result in useful molecular probes of BAP1 function and help to clarify the role of BAP1 in BRCA1 mediated events.